The role of phosphorylation in regulating the antidiabetic effects of O3FAR-1

描述（由申请人提供）：ω-3脂肪酸受体（O3 FAR 1），通常也称为GPR 120，是一种G蛋白偶联受体（GPCR），已显示其被长链不饱和脂肪酸（特别是属于ω-3脂肪酸（O3 FA）家族的那些）激动。这种受体由于其刺激肠促胰岛素激素胰高血糖素样肽-1（GLP-1）的肠分泌的能力而引起了相当大的兴趣，GLP-1刺激胰腺b细胞，导致胰岛素分泌，随后降低血糖。除了这种作用之外，最近已经证明，03 FAR 1的激动通过抑制NF-κ B和JNK促炎途径导致深刻的抗炎作用，当在巨噬细胞中活化时，其导致胰岛素抵抗、体重增加和2型糖尿病。我们的实验室已经发现，两个人类O3 FAR 1亚型，O3 FAR 1长和短，存在作为替代mRNA剪接的结果，我们的研究结果表明，这些亚型的差异受受体磷酸化调节。我们的初步数据还表明，G蛋白偶联受体激酶-6（GRK 6）是负责同源（激动剂诱导）磷酸化O3 FAR 1亚型。重要的是，O3 FAR 1的抗糖尿病作用已被证明依赖于b-抑制蛋白-2伴侣蛋白，其仅被募集到GRK-磷酸化受体，表明受体磷酸化驱动O3 FAR 1抗糖尿病活性。此外，我们的研究结果表明，基础磷酸化的O3 FAR 1，调节受体的表达和激动剂的敏感性，是由蛋白激酶C（PKC）介导的，并有两个O3 FAR 1亚型的差异影响的PKC。我们提出了两个具体的目标，这将提供结构上的指导机制O3 FAR 1的激活和信号调节，也将提供一个更好的理解两个O3 FAR 1亚型之间的信号差异。在具体目标一中，我们将使用蛋白质组学驱动的方法定位GRK和PKC介导的O3 FAR 1亚型磷酸化的特异性位点。在具体目标二中，我们将评估这些磷酸化机制在调节受体与b-抑制蛋白-2相互作用、激活Gaq/11-磷脂酶C/Ca+2信号通路、分泌GLP-1以及通过抑制JNK和NF-κ B通路产生抗炎作用方面的功能意义。总的来说，这些结果将提供结构洞察所有已知的O3 FAR 1功能的磷酸调节机制。由于在药学和健康科学的美世大学的学院的研究活动是完全依赖于学生的整合，该项目将允许研究生（博士），专业（PharmD），并在一个既定的和重要的研究项目本科生的合并，并将大大加强研究环境，与区域资助机制的目标一致。

DESCRIPTION (provided by applicant): The omega-3 fatty acid receptor (O3FAR1), also commonly referred to as GPR120, is a G protein-coupled receptor (GPCR) that has been shown to be agonized by long chained unsaturated fatty acids, specifically, those belonging to the omega-3 fatty acid (O3FA) family. This receptor has generated considerable interest due to its ability to stimulate intestinal secretion of the incretin hormone glucagon-like-peptide-1 (GLP-1), which stimulates pancreatic b-cells leading to insulin secretion and subsequently, decreases in blood glucose. In addition to this effect, it has recently been demonstrated that agonism of O3FAR1 leads to profound anti- inflammatory effects via inhibition of the NF-KB and JNK proinflammatory pathways, which when otherwise activated in macrophages, lead to insulin resistance, weight gain, and type 2 diabetes. Our laboratory has revealed that two human O3FAR1 isoforms, O3FAR1-long and short, exist as a consequence of alternative mRNA splicing and our results suggest that these isoforms are differentially regulated by receptor phosphorylation. Our preliminary data also show that G protein-coupled receptor kinase-6 (GRK6) is responsible for homologous (agonist-induced) phosphorylation of O3FAR1 isoforms. Importantly, the antidiabetic effects of O3FAR1 have been shown to be dependent on b-arrestin-2 partner proteins, which are recruited only to GRK-phosphorylated receptors, suggesting that receptor phosphorylation drives O3FAR1 antidiabetic activity. Additionally, our results demonstrate that basal phosphorylation of O3FAR1, which regulates receptor expression and agonist sensitivity, is mediated by protein kinase C (PKC), and that there are differential affects of PKC on the two O3FAR1 isoforms. We propose two specific aims that will provide structural guidance on mechanisms of O3FAR1 activation and signal regulation, and will also provide a better understanding of signaling differences between the two O3FAR1 isoforms. In specific aim one, we will localize the specific sites of GRK and PKC mediated phosphorylation of O3FAR1 isoforms using a proteomic- driven approach. In specific aim two, we will assess the functional significance of these phosphorylation mechanisms with respect to their ability to regulate receptor interactions with b-arrestin-2, activate the Gaq/11- phospholipase C/Ca+2 signaling pathway, secrete GLP-1, and produce anti-inflammatory effects via inhibition of JNK and NF-KB pathways. Collectively, these results will provide structural insight into mechanisms of phospho-regulation of all known O3FAR1 functions. Since research activities at Mercer University's College of Pharmacy and Health Sciences are fully dependent on student integration, this project will allow for incorporation of graduate (PhD), professional (PharmD), and undergraduate students within an established and significant research project, and will greatly strengthen the research environment, consistent with the goals of the AREA funding mechanism.